The actin-activated MgATPase activity of Acanthamoeba myosin IC (AMIC)is regulated by phosphorylation of Ser329 in the catalytic domain. Last year, we showed that the S329E mutant is constitutively fully active and the S329A mutant is constitutively inactive. We now examined whether structural differences between the active and inactive forms could be discerned by cryoelectron microscopy. No differences were observed at 24 angstrom resolution between the reconstructed images of the rigor complexes of the two mutants bound to F-actin or between the rigor and ADP-states of the S329E mutant bound to F-actin. The catalytic domain of AMIC has the same shape and binds to F-actin at the same angle as was previously shown by others for the catalytic domain of AMIB, but the domain structure and orientation of the tails are different for the two isozymes. The full-length kinase (MIHCK) that phosphorylates serine-329 of AMIC (and related isozymes) and activates its actin-dependent ATPase activity has now been cloned and sequenced and found to contain a consensus p21-binding site in its N-terminal regulatory domain. We had previously shown that MIHCK is activated by acidic-lipid dependent autophosphorylation. We now find that, like mammalian PAKs, autophosphorylation of MIHCK is activated by the p21s Rac and Cdc42 (and not by Rho) but, in contrast to mammalian PAKs, this activation requires the presence of acidic lipids. The acidic lipid-binding site is near the N-terminus followed by the p21-binding region. This N-terminal regulatory domain contains strongly positive and strongly negative regions and the extremely Pro-rich middle region has a strongly acidic N-terminal segment and a strongly basic C-terminal segment. We propose that autophoshorylation activates MIHCK by neutralizing the basic segment of the Pro-rich region thus unfolding the regulatory domain and abolishing its inhibition of the catalytic domain. In addition to the studies descrbed above, we have prepared a number of truncated and deletion mutants of AMIC to use in studies of the structure of the AMIC tail, the precise regions required from binding to membranes and F-actin, and the regions that affect its affinity for MIHCK. Expression of full-length MIHCK is being pursued as an essential step in determining its autophosphorylation sites, p21-binding sites, acid lipid-binding sites, etc. and the regions involved in the interaction of MIHCK with its substrate, AMIC. Also, studies of the conformational changes that regulate the activity of myosin II when the tip of the tail is phosphorylated are continuing through the use of expressed wildtype and mutant rods.
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